The present invention relates to the fields of molecular biology and immunology. More specifically, this invention relates to the induction of apoptosis in inflammatory cells by introducing into those cells a gene which induces apoptosis (programmed cell death or non-necrotic cell death) in these cells.
In many inflammatory conditions, cytokines such as IL-1xcex2, IL-10, GM-CSF and TNFxcex1 are excessively produced as a result of mass aggregation and accumulation of inflammatory cells (Brennan F. M. et al., British Medical Bulletin 1995, 51/2, 368-384). Upregulation and/or dysregulation of cytokines in inflamed tissue may be directly or indirectly responsible for exacerbation of chronic inflammatory diseases. For example, the most marked pathology in rheumatoid arthritis (RA) is displayed at the local site of inflammation (i.e., the synovial joints). Therefore, it is likely that the cytokines produced in the synovial joints of RA patients play an important role in the disease process. Of those cytokines, IL-1xcex2 and TNFxcex1 are believed to be responsible for the devastating cartilage destruction and bone erosion which characterizes RA (Dayer J. M. et al., J. Exp. Med., 1985, 162, 1208-1215; Gowen M. et al., Nature, 1983, 306, 378-380). The presence of excessive amounts of IL-1xcex2 and TNFxcex1 in the synovial joints has been shown to accelerate development of collagen-induced arthritis in rodents (Brennan F. M., et al., Clin. Expt. Immunol., 1994, 97/1, 1-3).
Apoptosis is a fundamental physiological process for embryonic development and maintenance of tissue homeostasis. (Raff, M. C. Nature, 1992, 356, 397; Vaux, D. L. et al. Cell, 1994, 76, 777). Inconsistency in this critical natural process is featured in a variety of neoplastic, neurodegenerative and autoimmune diseases (Thompson, C. B., Science, 1995, 267, 1456). Biochemical attributes, involving signal transduction cascade, are relatively complex and are not completely understood. A variety of stimuli including activation of specific receptors, such as TNFR1 or Fas trigger evolutionary conserved execution machinery involving several signaling components, that are orchestrated to cause cellular demise (Ashkenazi, A. and Dixit, V. M., Science, 1998, 181, 1305).
Granzyme B is a serine protease, primarily found in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. Granzyme B plays an important role in inducing apoptotic changes in target cells by cytotoxic cell mediated killing (Huesel J. W. et al, Cell, 76, 977-987, 1994; Shi, L. et al. J. Exp. Med. 176,1521-1529, 1992), partly by catalyzing cleavage and activation of several caspases (Salvesen, G. S. and Dixit, V. M., Cell, 91, 443-446, 1997) as well as by caspase independent pathways (Andrade, F. et al. Immunity 8,451-460,1998). Structurally, Granzyme B is produced as polypeptide containing a leader peptide separated by an inactivating di-peptide (Gly-Glu) from the active Granzyme B polypeptide. Like the caspases, Granzyme B recognizes substrates specifically at aspartic acid for cleavage.
TNFxcex1 is a cytokine, mainly synthesized by monocytes, macrophages and lymphocytes in response to activation. The classic elements governing its expression are located in the proximal or the distal promoter region (reviewed in Pauli, U. Critical Rev. in Eukaryotic Gene Expression, 1994, 4, 323-344). Summarized below are regions that have been described to play a significant role in the TNFxcex1 promoter activity:
a) TNFxcex1-responsive elements were shown to be located between base pairs xe2x88x92100 to xe2x88x92125. The region xe2x88x92108 to xe2x88x92101bp contains a palindrome, TGAGCTCA , which is similar to AP-1 sequence that contains PMA-responsive elements. Multiple copies of xe2x88x92125 to xe2x88x9285bp confer a 7 to 11 fold induction of the expression of the reporter gene (Leitman, D. et al, J. Biol. Chem. 266, 9343, 1991).
b) PMA-responsive elements were shown to be present in between xe2x88x92101 to xe2x88x92286 base pairs (Hensel, G. et al, Lymphokine Res. 8, 347, 1989).
c) Anti-CD3 antibody-induced (as well as Ca-ionophore- induced) responsive elements were shown to lie between xe2x88x92118 to xe2x88x9280 base pairs. The KappaB3 (GGGTTTCTCC) SEQ ID NO: 15 sequence in this region is of high importance for CsA-sensitive activation of the TNFxcex1 promoter by Ca-ionophore. These elements are suggested to be optimally functional in the context of their own promoter (Goldfield, et al. J. Exp. Med. 178, 1356, 1993).
d) In U937 cells, the PMA responsive element is located between xe2x88x9295 to xe2x88x9236bp and the cAMP-responsive element (CRE) is mapped to position xe2x88x92107 to-99bp. This region does not respond to PMA (Economou, J. S. et al , J. Exp. Med. 170, 321, 1989).
e) All three kappaB sites [viz. kappaB1 (xe2x88x92587 to xe2x88x92577), kappaB2 (xe2x88x92210 to xe2x88x92202) and kappaB3 (xe2x88x9298 to xe2x88x9287) ]bound virus-inducible protein, although deletion of these sites did not affect virus inducibility (Goldfield, A. et al, PNAS, 87, 9769, 1990). Further more, deletion mutants of kappaB sites show that they are not primary targets for PMA stimulation of human TNFxcex1 gene (Goldfield, A. et al J. Exp. Med. 174, 73, 1991).
f) In the murine system, the TNFxcex1 promoter constructs xe2x88x921059, xe2x88x92695 and xe2x88x92655bp are strongly LPS inducible. This LPS-inducibility was greatly reduced in a xe2x88x92451bp construct and further between xe2x88x92301 and xe2x88x92241bp. The xe2x88x921059bp fragment of TNFxcex1 promoter was silent in macrophages and was strongly expressed after LPS stimulation. The largest drop of activation was at xe2x88x92695 to xe2x88x92655bp, which contains a kappaB element in the murine TNFxcex1 promoter (Shakhov, A. N. et al, J. Exp. Med., 1990, 171,35; Drouet, C. et al, J. Immunol., 1991, 147, 1694).
Elements in the 3xe2x80x2 untranslated region (3xe2x80x2UTR) of the TNFxcex1 gene are known to be important for post-transcriptional regulation. Analysis of the influence of 3xe2x80x2 UTR has been made in the murine system, wherein conjunction with the homologous promoter, LPS inducibility was very strong. Using murine TNFxcex1 promoter system, it was shown that 3xe2x80x2UTR effectively inhibits CAT activity in three non-macrophage cell lines viz. HeLa, NIH3T3 and L929. The sequence TTATTTAT was repeated several times in the 3xe2x80x2UTR and was proposed to be involved in regulation (Han, J., et al., J. Immunology, 1991, 146, 1843-1848; Crawford, F. K., et al., J. Biol. Chem., 1996, 271, 22383-22390).
A variety of cells such as activated macrophages, activated T cells, macrophage-like synoviocytes as well as fibroblasts-like synoviocytes, and transformed macrophage-like synoviocytes (also referred to as pannocyte) are present in the inflamed joints. An invasive structure, called the pannus, derives from the hyperplastic nature of synoviocytes and pannocytes. The pannus may result from an excessive proliferation of cells and/or diminished apoptosis in these cells. Proliferative index in these cells was shown to be relatively low. Therefore, hyperplasia in synoviocytes could be due to abnormalities in apoptosis of the synovial lining. The frequency of cells with end stage apoptosis is low in the synovium. Abnormalities of p53 mutations, which could result in resistance to apoptosis are reported in the RA synovial fibroblasts. Additionally, excessive amounts of pro-inflammatory cytokines such as TNFxcex1 and IL-1xcex2 are produced in the synovial tissue by a variety of cell types at the cartilage-pannus junction, including cells of the macrophage lineage, macrophage-like synoviocytes, activated T-cells and possibly fibroblast-like synoviocytes (Chu C. Q. et al., Arthritis and Rheumatism, 1991, 34, 1125-1132; Deleuran B. W., et al., Arthritis and Rheumatism, 1992, 35, 1170-1178). This perpetuates the infiltration of inflammatory cells and production of more pro-inflammatory cytokines and factors, which are responsible for synovial cell proliferation. In addition to the above described inflammatory effects, TNFxcex1 plays a ubiquitous and key role in a variety of pro-inflammatory events.
TNFxcex1 induces IL-1xcex2 activity in monocytes. Indeed, anti-TNFxcex1 neutralizing antibodies have been shown to reduce overall IL-1xcex2 production (Portillo, et al., Immunol., 1989, 66, 170-175; Brennan F. M., et al., British Medical Bulletin 1995, 51/2, 368-384). Thus, an added benefit to blocking the effect of the inflammatory cytokine TNFxcex1 was the reduction in production of the equally destructive pro-inflammatory mediator, IL-xcex2. Furthermore, it is well known that TNFxcex1 is a transcriptional activator of other inflammation-related genes. For example, the presence of TNFxcex1 stimulates production of other cytokines (such as GM-CSF) and cell surface receptors, including HLA class II antigens and adhesion molecules (Alvaro-Garcia J. M., et al., J. Exp. Med., 1989, 146, 865-875), that perpetuate recruitment of activated T cells and neutrophils resulting in synovial inflammation and hyperplasia and ultimately, in augmented destruction of cartilage and bone (Allen J. B., J. Exp. Med., 1990, 171, 231).
Conventional therapy against inflammatory disorders is typically directed against symptomatic inflammation. Such therapy provides only temporary relief without significantly delaying disease progression. In contrast, therapies targeting TNFxcex1 and other factors induced in the inflammatory process are likely to be more promising. For example, in collagen-induced arthritis animal models, an anti-TNFxcex1 antibody and soluble TNFxcex1 receptor-IgG chimera effectively reduced paw swelling, joint involvement and cartilage and bone destruction (Williams R. O. et al., Proc. Natl. Acad. Sci., 1992, 89, 9784-9788). Human trials using both humanized anti-TNFxcex1 antibodies and TNFxcex1 receptor-IgG chimeric molecules produced dramatic results (Elliott M. J., et al., Arthritis and Rheumatism, 1993, 36, 1681-1690; Elliott M. J., et al., Lancet, 343, 1105-1110). Although treatment with these TNFxcex1 antagonists appears to be well tolerated, it also results in production of antibodies against the recombinant proteins. Thus, these therapies may not be suitable for long term treatment and do not achieve true disease abatement.
WO 97/07828 discloses methods of treating by gene therapy a patient with cellular accumulation or a chronic inflammatory disease which was a result of a defective apoptosis-regulating gene, more specifically p53. The treatment restores the defect with a wild-type gene attached to a promoter which drives the apoptosis- regulating gene expression.
In order to actually modify progression of the disease, TNFxcex1 must be continuously targeted using TNFxcex1-specific therapies. Such a continuous therapeutic protocol was impractical with these biologic agents and would be difficult to administer in the long term.
In an alternate therapeutic option, inflamed synovium may be removed using surgical (Herold N. and Schroder H. A., Acta Orthop. Scand., 1995, 66, 252-254; Ogilvie-Harris D. J. and Weisleder L., Arthroscopy, 1995, 11, 91-95), chemical (Cruz-Esteban C. and Wilke W. S., Bailliere""s Clinical Rheumatol., 1995, 9, 787-801) or radiation-induced synovectomy (Cruz-Esteban C. and Wilke W. S., Bailliere""s Clinical Rheumatol., 1995, 9, 787-801). Marginal to good results follow arthroscopic surgery. Non-surgical synovectomy is performed using various chemical agents such as osmic acid, alkylating agents such as nitrogen mustard and thiotepa, methotrexate. Unfortunately, non-surgical synovectomies (including chemical and radiation-induced) are procedurally complicated, provide only short term relief and show only patchy reduction of the synovial hyperplasia. Furthermore, most of the non-surgical alternatives are potential teratogens. Moreover, an innate inflammatory response is concomitant with tissue damage arising from the chemical or surgical intervention. Finally, it should be noted that these approaches suffer from the risks and side-effects commonly associated with conventional pharmaceutical therapy and invasive surgical procedures, including the expense and inconvenience of hospitalization and rehabilitation.
Accordingly, a need still exists for an effective therapeutic approach to treating inflammatory disorders in general and RA in particular.
The invention provides methods and compositions relating to a novel apoptosis induced destruction of TNFxcex1 producing cells. It was therefore an object of the invention to provide unique chimeric nucleic acid molecules having at least one TNFxcex1 promoter enhancer region (comprising the nucleic acid SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 12 or conservative substitution or allelic variants thereof) attached to a TNFxcex1 promoter, the TNFxcex1 promoter further being attached to a nucleic acid sequence encoding the Granzyme B protein or conservative substitution or allelic variants thereof which in turn was further attached to a 3xe2x80x2UTR nucleic acid sequence.
Another objective of this invention was to provide TNFp-AIG and the like chimeric nucleic acid constructs, processes for making them, methods of using them, and preparations containing them.
It was a further object to provide a method of treating an inflammatory disorder by administering to a patient in need of such treatment a pharmaceutically effective amount of the composition having the chimeric nucleic acid molecule described herein.
It was yet a further objective of this invention to provide a method for the induction of apoptosis in cells transfected with the TNFp-AIG chimeric nucleic acid, a method for the in vitro selection of TNFxcex1 non-producer somatic cell variants in a population, a method for identifying dominant/negative genes responsible for the genesis of a TNFxcex1 non-producing population and a method for identifying products responsible for regulation of TNFxcex1 production.
These and other objectives will be readily appreciated by those of ordinary skill in the art based upon the following detailed disclosure of the invention.